-1-
Introduction
The high intertidal gastropod Littorina planaxis
excretes a variety of nitrogenous waste products (Campbell,
1970; Cox, 1964; Duerr, 1968; Needham, 1938). In the
kidney, ammonia is the predominant non-protein nitrogenous
waste product (Campbell, 1970; Cox, 1964). Cox found
evidence of a tidal rhythmicity in the amount of ammonia
found in the kidney. The highest concentrations of
ammonia were found during the ascending tides. After
this point, the amount decreased, reaching a minimum
value at low tide. While small amounts of ammonia may
be liberated as gas when the animals are dry (Lebenzon,
1964), Duerr (1968) found that several species of marine
prosobranch molluscs, including Littorina sitkana, excrete
ammonia in solution upon being immersed. Immersion may
stimulate excretion. Kops (1964) found that moisture
was a stimulus to activity. While Kops found no evidence
of a circadian effect on activity, observations indicate
that physical activity does increase at night, and an in¬
crease in metabolic and/or excretory activity may parallel
this increase. If moisture is indeed a stimulus for ac¬
tivity and for excretion, L. planaxis may be exploiting
two periods of high moisture. The rocky substrate on
which it lives is moistened by the splash of the high
tide and by dew which condenses during the night. Fluc¬
tuations in ammonia excretion during the tidal and diurnal
-2-
cycles are the focus of the study reported here.
Materials and Methods
Snails were collected from the rocky intertidal at
the Hopkins Marine Station, Pacific Grove, California.
The vertical range of the collecting site was 8 to 12 feet
above mean lower low water. Ammonia was collected from
five replicates of ten snails each, which were placed in
250 ml. beakers with 5 ml. sea water and swirled, to wet
the snails thoroughly. After one hour, the beakers were
swirled again to mix the sample, the snails were removed,
and the water was centrifuged to remove fecal material.
One ml. aliquots were removed and tested for the presence
of ammonia using a colorimetric proceedure (Ternberg and
Hershey, 1964). After color development, centrifugation
was necessary to remove precipitated carbonates. Samples
were read in a Gilson 252 Spectrophotometer at 625 nm,
against a distilled water blank, and corrected for any
ammonia present in the sea water used for collecting
ammonia.
Duerr (1968) found a correlation between the amount
of ammonia excreted and the total weight of the animal.
The amounts of excreted ammonia were normalized by dividing
by the weight of the snails sampled, and expressed as
ug ammonia/ g snail.
Results
The fluctuations in ammonia excretion by animals
during the tidal and diurnal cycles were studied as follows.
Snails were collected from the field at the high and
low tides and at times intermediate between these extreme
points. Snails were brought into the laboratory and
tested within fifteen minutes. Figure 1 shows the results
of this study. Note the wide range of amounts of ammonia
excreted at different times. Individual values range from
.65 to 3.05 ug NH3/ g snail, and the means range from
1.15 to 2.60 ug NH3/ g snail. The lowest individual
value and the lowest mean value both occur during the day¬
time low tide, while the highest individual value and the
highest mean value both occur at high tide after sunset.
The lowest values obtained during the night, during the
falling and low tides, have means 22% and 61% higher, respec¬
tively, than means for similar points on the tidal cycle
during the day. During the night, excretory activity in¬
creases throughout the tidal cycle.
A similar study was made using the same five groups
of ten snails at each sample time in an attempt to decrease
the variability observed in the previous study. Fifty
snails were collected at 0900 hours, on the ascending tide.
Between sampling times, the snails were left outside the
laboratory, subjected to the normal diurnal regime, and
brought into the laboratory only for testing. This ex¬
periment eliminated any tidal effect, because the snails
were immersed every three hours. Figure 2 presents the
results. Figure 3 shows the amount excreted by the in¬
dividual test groups.
Again, the lowest values are obtained during the
daylight hours; values rose as night approached and
decreased again after sunrise. Variability did decrease;
the 95% confidence intervals ranged from +.05 to +.50 ug NH3/
g snail, while the 95% confidence intervals for values
obtained during the field study ranged from +.15 to +.90
Ug NH3/g snail. The snails excreted an average of 40%
more ammonia the next morning, after twenty-four hours
of periodic immersion. After the final test at 1400 hours,
the snails were again placed outside and tested after
twenty hours of dryness. The mean value, .03 ug NHa/g snail,
decreased to the level obtained at the start of the first
day, when the snails had been collected on a low tide.
Since a period of dryness appears to depress excretory
activity, the next study examined the effects of prolonged
dryness, Figure 4. Four hundred snails were collected at
1330 hours, on the descending tide. The snails were
kept in an open container outside, subject to the normal
light regime, to normal temperature fluctuations and to
deposition of dew, yet were removed from the beach area,
eliminating any exogenous tidal influences. At every
test time, fifty fresh snails were taken from the con¬
tainer and brought into the laboratory for testing. The
-5
large rise in amount of ammonia excreted which was evi¬
dent in the previous two studies is not apparent. The
range of means is much narrower, .65 to 1.10 ug NH3/g snail.
The smallest 95% confidence interval occurs at the low
tide, 1600 hours, only two hours after collection. As
the tide rises, yet the period of dryness increases, var¬
iability increases, except for the point at 0830, on the
ascending tide just after sunrise. The lowest mean occurs
at 1200 hours, the final testing point, at the high tide.
Under abnormal moisture conditions, the snails display a
high degree of individual variability. Some may have been
affected more by the lack of moisture and excreted less, or
not at all.
A fourth experiment, shown in Figures 5 and 6, ex¬
amined differences in excretory behavior between snails
kept in constant light or constant dark. One hundred
snails were collected from the field at 0900 hours, on
the descending tide. Fifty snails, in five groups of ten
each, were placed in constant dark and fifty were placed
in constant light. The first sample was tested at 1100
hours, during the low tide, so that the snails in the
dark had two hours to respond to the change in light
conditions. The same ten groups, five in constant arti¬
ficial light and five in constant dark, were sampled at
each test time. Both groups showed peaks of excretion at
the high tide; the snails kept in constant light showed
a larger difference in amplitude between day and night peaks
than those kept in the dark. During hours of normal daylight,
-6-
the 95% confidence interval of snails kept in the light
was similar to that of snails periodically immersed yet
kept on a normal day/night cycle, see Figure 2. After the
time of sunset, however, the 95% confidence intervals
increased, becoming almost three times that of snails in
Figure 2 at 0400 hours. The snails kept in constant dark
had consistently larger confidence intervals than those
of Figure 2, increasing up to 188% at 0000 hours. Again,
under abnormal conditions, the snails display a greater
degree of individual variability.
Another set of investigations attempted to discover
if any relationship exists between amounts of ammonia syn¬
thesized and time of day. At 1100 and 2100 hours, the standard
sampling proceedure was followed, although sample size was
increased to twenty snails and 10 ml. of sea water was
used. At fifteen minute intervals, samples were withdrawn
and fresh sea water was added to maintain a constant vol¬
ume. The values for subsequent readings were corrected for
this dilution effect. Figure 7 shows the difference
between two groups of snails, one from each testing period.
Initially, the amounts of ammonia excreted by the two groups
appear to be identical. However, during the night,
amounts excreted increase more than during the day.
Discussion
The presence or absence of light seems to be the
-7-
major factor in determing how much ammonia L. planaxis
excretes. However, in the absence of light and dark cues,
the tidal correlations become more apparent. In Figure 1,
this combined diel/tidal cycle is most obvious; since
snails were collected from the field at every test point,
they were subject to normal environmental conditions until
just prior to testing, i.e., they experienced up to seven
hours between times of maximum moisture at the high tides.
The amounts of excreted ammonia decrease after sunrise,
and the final amount at 0900 parallels the amount found at
the same time on the previous day. This may indicate that
uniformly low levels of ammonia are excreted during the
morning hours; the possibility also exists that the snails
collected at this time, which coincided with high tide,
had already excreted on the rising tide or just prior to
the peak of the tide.
In contrast, the groups of snails which were immersed
every three hours excreted larger amounts of ammonia on
the second morning, Figures 2 and 3. In all cases, however
the snails excreted less on the third morning, after twen¬
ty hours of dryness. Moisture does seem to stimulate the
snails to excrete. All individuals exhibit a diurnal pat¬
tern of excretion; excretory activity increases after
sunset and decreases after sunrise. Whether the absense
of tidal correlations is due to periodic immersion or to
the coincidence of evening with the high tide cannot be
determined; repeated studies during as evening low tide
might reveal the degree of tidal influence.
The hypothesis that moisture is a stimulus to excrete
is supported by the data shown in Figure 4. The values
obtained at the first sample time, 1400 hours, are almost
identical to those in Figure 1, from snails brought in
from the field and tested at 1400 hours. However, these
snails fail to show any large increase in the amount
excreted, despite the onset of darkness. While mean values
for snails taken from the field range from 1.15 to 2.60
Ug NHa/ g snail, the mean values for the snails which
were kept dry only range from .65 to 1.30 ug NH3/g snail.
The small 95% confidence interval at 1600 hours, during
low tide, may be due to the fact that, at this time,
experimental conditions most nearly paralleled conditions
in the field. As the tide rises, yet the snails exper¬
ience no increase in moisture, there is no large increase
in mean values, while 95% confidence intervals increase.
The snails are differentially affected by the absence of
moisture. The interval is again small at 0830 hours;
this test period occurs after sunrise, on the ascending
tide. Moisture from dew had been observed in the container;
perhaps, if the snails are indeed on a tidal cycle, this
moisture signifies an incoming tide to some, which, an¬
ticipating a return of normal conditions, begin to excrete
at a normal level. The mean value here, 1.20 +.20 ug NH./
g snail, is quite similar to that found in the field study
at 0830, 1.20 +.45 ug NH3/g snail. The value at 1200
-9
decreased, however, despite the occurrence of high tide;
two factors may account for this. Firstly, a similarly
low level of excretory activity is displayed by snails
taken from the field, in Figures 2 and 5; however, these
low levels coincide with the low tide. Secondly, if
the above hypothesis describing dew as a false moisture
cue is correct, many of the snails may have excreted
during what they assumed would be an ascending tide; yet,
at high tide, when no moisture was forthcoming, some did
not excrete, or excreted less. Such a conjecture is sup¬
ported by the findings graphed in Figures 2 and 3, where
snails kept dry for twenty hours after twenty-eight hours
of testing excreted less than they had at the same time
on the previous day. Due to the large confidence inter¬
vals and the small range of means, Figure 4 does not in¬
dicate that any appreciable correlation exists between the
amount excreted and either light or tidal conditions.
While a large measure of variability may be due to dif¬
ferences among the five groups, i.e., different groups
of fifty snails were tested at each dampling time, this
graph may also indicate that, deprived of moisture, L.
planaxis has less ammonia available to excrete.
If the snails are given what appears to be an op¬
portunity to continue to make and excrete ammonia, i.e.,
if they are periodically immersed as those in Figures 2
and 3 were, test conditions of constant light and constant
dark might obliterate the diurnal correlations observed
-10-
in Figures 2 and 3. In Figure 5, the mean values of
both the groups kept in constant light and those kept
in constant dark parallel the tidal cycle. The smaller
amplitude of the daytime peak of the snails kept in
constant light is consistent with results indicating
that less ammonia is excreted during the day. The maxi¬
mum nighttime values, at the peak of high tide, are al¬
most identical to those of snails kept in constant dark¬
ness, and are greater than those obtained from the snails
described in Figure 2. This suggests that, at least during
the first day of constant light, an endogenous rhythmicity
in excretory activity is not damped out. The snails are
perhaps differentially affected by the abnormal conditions,
however; while these five groups has 95% confidence inter¬
vals of +.20 to +.40 ug NH3/ g snail during normal hours
of daylight, the confidence intervals increase to +.65
to +1.10 ug NHa/ g snail after the time of sunset. The
snails placed in constant darkness always displayed
high variability; once more, some may have been affected
more than others by the abnormal conditions. The constant
dark groups, also paralleling the tidal cycle, gave higher
values during daylight hours than the snails kept in con¬
stant light; the artificial onset of darkness seems to
be a cue to increase ammonia excretion. Following the
patterns of individual groups, Figure 6, groups 1-8 parallel
both tidal cycles. The constant light groups 3, 4 and 5
-11-
seem to anticipate the high tide which occurs at sunrise.
Two possible explanations of this behavior exist. The
snails, deprived of normal light/dark cues, may have mis¬
calculated the time of high tide; since high tide occured
at sunrise, the snails would be expected to excrete rela¬
tively large amounts at the onset of light, yet they
receive no change in light conditions which might cue
their response. On the other hand, prior to this time,
none of the groups excrete large amounts which would
normally be excreted during hours of darkness; perhaps
they are storing ammonia until internal concentrations reach
levels which they can no longer tolerate, and they excrete,
regardless of time of day or point in the tidal cycle.
The constant dark groups 9 and 10 did not show an increase
and decrease in values during the daytime high tide. Per¬
haps the sudden advent of darkness failed to elicit the
increase in amount excreted observed in the other two
groups. Note, however, that sunset coincides with the
falling tide on this day; the snails may have been res¬
ponding to what appeared to be sunset, and excreted smaller
amounts of ammonia until the tide began to rise, after
0300 hours. The premature onset of darkness does not,
however, seem to affect the tidal pattern of excretion
beyond the first tidal cycle. Further investigations of
levels of excretory activity under prolonged constant
light and constant dark regimes might elucidate the nature
-12-
of this excretory rhythmicity, i.e., whether it is truly
endogenous or rather a response to external conditions,
Thus, while light conditions appear to be the strongest
factor influencing ammonia excretion, the absence of light
and dark cues make tidal correlations more apparent.
Given the high intertidal habitat of L. planaxis, which
has a vertical distribution of 5 to 12 feet above mean
lower low water (Peterson, 1964), night appears to be
a more reliable moisture cue; while dew is usually deposited
on the rocks after dark, the snails may not always be splashed
by the high tide, depending in their position on the rocks
and the height of the surf.
Note that, while in previous tests, Figures 2,3 and 4,
the absence of moisture seems to inhibit ammonia excretion,
the periodic wetting of the snails, which eliminates
normal tidal cues, does not appear to damp out variations
in levels of excretion apparently correlated with the tide.
Cox (1964) found the lowest amount of ammonia in the kid¬
neys at lower low water, the period of maximum dryness;
this finding corroborates the results which indicate that
snails which are kept dry excrete less ammonia. Perhaps
moisture is not only a stimulus for excretion, but,
under conditions of periodic moisture, the snail has more
ammonia available to excrete. Cox also found that the
kidneys contained the greatest amount of ammonia on the
ascending tide. The snails have been shown to excrete the
largest amounts of ammonia on the ascending and high
-13
tides. If the ammonia synthesized and stored in the kidney
were excreted during this period, the amount in the
kidney would be less at high tide than during the ascending
tide. Moisture would facilitate ammonia excretion, since
ammonia is soluble in water. Because large concentrations
of ammonia are toxic, storing quantities of ammonia for
extended periods of time might be harmful to the animal;
thus the snails have ammonia available to excrete only
during those times at which they are assured of the avail¬
ability of moisture, i.e., at night, when dew falls, and
at the high tides.
This hypothesis indicated that rates of synthesis,
as well as amounts of available excretory products, may
vary. Preliminary studies found that, over a four hour
period of testing, the test water contained increasing
amounts of ammonia for the first one to two hours, yet,
after two hours, the concentration of ammonia in the test
water remained fairly constant during the day. However,
during the night, the concentration of ammonia in the test
water increased over the entire four hour test period.
During the day, the snails, in the first one to two hours
of immersion, may have excreted any ammonia which they had
accumulated. After this point, levels of excretory products
would increase only slightly, if at all, if the rate of
synthesis is low. At night, however, the amount excreted
would continue to increase to a greater extent if the rate
of synthesis is appreciably higher. Figure 7 gives data
-14-
for two different groups of twenty snails each, one tested
during the morning and the other tested in the evening,
while kept in constant dark. These two groups show striking
differences in excretory behavior. While the initial
excretory rates of the two groups seem comparable, the
snails tested during the day seem to synthesize and excrete
a smaller amount of ammonia than those tested at night.
The effect of tide on rates of excretion remains to be
determined.
-15
Summary
1) Moisture appears to be a stimulus for ammonia
excretion; since this nitrogenous waste product is water¬
soluble, ambient moisture would facilitate diffusion.
2) L. planaxis displays peaks of excretory activity
which coincide with times of greatest moisture, i.e.,
night and high tide, thus maximizing opportunities for
excretion.
3) Light and dark seem to be the strongest factors
influencing this change in excretory activity; the tidal
correlations become more apparent when normal light and
dark cues are removed.
4) If deprived of moisture, L. planaxis excretes
smaller amounts of ammonia; since this compound is toxic,
ammonia synthesis could be harmful if no means were avail¬
able for excretion.
5) Rates of ammonia synthesis appear to be greater
at night than during the day.
Acknowledgements
I would like to thank Dr. John H. Phillips for his
insights, suggestions and encouraging winks.
-16
Literature cited
Campbell, J. W. and Bishop, S. H. (1970), Nitrogen meta¬
bolism in molluscs in Comparative Biochemistry of
Nitrogen Metabolism, J. W. Campbell, ed., Academic
Press, London and New York, Vol. I, pp. 103-206.
A., (1964),An analysis of nitrogenous waste products
Cox,
in Littorina planaxis (unpublished manuscript on
file at the Hopkins Marine Station Library, Pacific
Grove, California.)
Duerr, F., (1968), Excretion of ammonia and urea in
seven species of marine prosobranch molluscs, Comp.
Biochem. Physiol. 26:1051.
E., (1964), The effect of certain environmental
Kops,
factors on the activity pattern of Littorina planaxis
and Littorina scutulata (unpublished manuscript on
file at the Hopkins Marine Station Library, Pacific
Grove, California.)
Lebenson, J., (1964), Respiration in Littorina planaxis
and Littorina scutulata (unpublished manuscript on
file at the Hopkins Marine Station Library, Pacific
Grove, California.)
Pederson, R. E., (1964) The lower limits of the habitats
of Littorina scutulata and Littorina planaxis (unpub-
lished manuscript on file at the Hopkins Marine
Station Library, Pacific Grove, California.)
Ternberg, J. L. and Hershey, F. B., (1964), Colorimetric
determination of blood ammonia, J. Lab. and Clin.
Med. 56:766-776.
Figure 1: Amounts of ammonia excreted by samples of
ten snails collected from the intertidal at
the times indicated. The open circles repre¬
sent the mean values, the bars represent 95%
confidence intervals and the closed circles
represent the actual values of each of the
samples.
U9. NH79 3I
88586 8
•
-


• —
—
— kk
—
—

tide
sunsel
sunrise
Figure 2: Amounts of ammonia excreted by samples of ten
snails kept in normal light conditions and
periodically tested. The open circles repre¬
sent the mean values and the bars represent
95% confidence intervals.
E9 NH/9 snail
8.
8
3
-e
—
—
—
—
k k
t-
tide
sundel
sungise
e
Figure 3: Amounts of ammonia excreted by the individual
groups of snails represented in Figure 2. Open
circles at 1100
hours indicate the amounts ex¬
creted after twenty hours of dryness.
(No data for groups 2 and 5 at 2200 hours.,
10

00
O

5 00


10

0


—
00
o

10

—8
00
10 12 14 16 18 20 22 0 2 4 6 8 10 12 14
lime oi doy
Figure 4: Amounts of ammonia excreted by samples of
ten snails kept in normal light conditions
and kept dry until time of testing. Open circles
represent the mean values, bars represent the
95% confidence intervals and closed squares
represent the actual values of each of the
samples.
28
5
19 NH3/g snail
8 56 8 8 8
—
—
—
k

— k
s

fide
unset)
Munset
Figure 5:
Amounts of ammonia excreted by samples of ten
snails kept in constant light or constant
dark and periodically tested. Open circles
represent the mean values and bars represent
the 95% confidence intervals.
35
30
20
15
10
5 05
oo
35
o0
10 12 14 16 18 20 22
O 2 4 | 6 | 8 10
imeoday
Figure 6: Amounts of ammonia excreted by the individual
groups of snails represented in Figure 5.
(No data for group 5 at 0600 hours, or for
group 7 at 2330 hours.)
.
2S
3
S
8 8 8 8
9 NH9 sni
88
tide
sunset
sunride
sunsel
sunrige
3L

dnoib
Figure 7: Amounts of ammonia excreted by samples of
twenty snails at the int
als indicate
Open circles indicate the amount excreted
by one sample group during the day. Open
squares indicate the amount excreted by one
sample group during the night.
Pis
a5
0



O
time (hours)